上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2022 , Vol. 42 >Issue 9: 1247 - 1257

DOI: https://doi.org/10.3969/j.issn.1674-8115.2022.09.011

论著 · 基础研究

敲减TCF3抑制人子宫内膜基质细胞蜕膜化的研究

  • 卫晓薇 ,
  • 田福举 ,
  • 刘晓瑞 ,
  • 曾维宏 ,
  • 陈彩莲 ,
  • 林羿
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  • 1.上海交通大学医学院附属国际和平妇幼保健院中心实验室，上海市胚胎源性疾病重点实验室，上海交通大学医学院出生缺陷与罕见病临床研究院，上海 200030
    2.上海交通大学电子信息与电子工程学院自动化系，系统控制与信息处理教育部重点实验室，上海 200240
卫晓薇（1996—），女，硕士生；电子信箱：kl_1996_kl@163.com。
林　羿，电子信箱：yilinonline@126.com。

收稿日期: 2022-05-19

  录用日期: 2022-08-29

  网络出版日期: 2022-09-28

基金资助

国家重点研发计划(2018YFC1002800);国家自然科学基金(82171669);上海交通大学“交大之星”重大项目(20210201)

收起

TCF3 knockdown inhibits the decidualization of human endometrial stromal cells

  • Xiaowei WEI ,
  • Fuju TIAN ,
  • Xiaorui LIU ,
  • Weihong ZENG ,
  • Cailian CHEN ,
  • Yi LIN
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  • 1.Central Laboratory of The International Peace Maternity & Child Health Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Key Laboratory of Embryo Original Diseases; Institute of Birth Defects and Rare Diseases, Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China
    2.Department of Automation, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University; Key Laboratory of System Control and Information Processing, Ministry of Education, Shanghai 200240, China
LIN Yi, E-mail: yilinonline@126.com.

Received date: 2022-05-19

  Accepted date: 2022-08-29

  Online published: 2022-09-28

Supported by

National Key Research and Development Program of China(2018YFC1002800);National Natural Science Foundation of China(82171669);Shanghai Jiao Tong University Trans-Med Awards Research (Major Project)(20210201)

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摘要

目的·探讨转录因子3（transcription factor 3，TCF3）对人子宫内膜基质细胞（human endometrial stromal cell，HESC）蜕膜化功能的影响。方法·用8-溴腺苷3'，5'-环腺苷酸（8-bromoadenosine 3'，5'-cyclic adenosine monophosphate，8-Br-cAMP）和醋酸甲羟孕酮（medroxy progesterone acetate，MPA）方案在体外诱导HESC蜕膜化。用小干扰RNA（small interfering RNA，siRNA）沉默TCF3基因，实时荧光定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）和Western blotting验证TCF3的敲减效率。RT-qPCR和酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）检测蜕膜化标志物胰岛素样生长因子结合蛋白1（insulin like growth factor binding protein 1，IGFBP1）和催乳素（prolactin，PRL）的表达水平。Alexa Fluor标记的鬼笔环肽染色检测HESC蜕膜化后及敲减TCF3后细胞形态的变化。将HESC分为3组，分别为未蜕膜化+NC（转染对照siRNA）组、蜕膜化4 d+NC组、蜕膜化4 d+siTCF3（转染TCF3 siRNA）组，进行RNA测序；对测序结果进行聚类分析、京都基因与基因组数据库（Kyoto Encyclopedia of Genes and Genomes，KEGG）分析和基因集富集分析（gene set enrichment analysis，GSEA）以探究TCF3调节HESC蜕膜化功能的作用机制，并采用qPCR对测序结果进行验证。结果·在HESC蜕膜化2 d和4 d后，TCF3的表达水平逐渐升高。蜕膜化2 d和4 d，ELISA和RT-qPCR结果均显示敲减TCF3后蜕膜化标志物IGFBP1和PRL的表达水平较NC组下降。鬼笔环肽染色结果表明，蜕膜化后细胞从纤细长梭形结构转变为大而圆的细胞形态，而在敲减TCF3后，细胞形态又恢复成长梭形结构。测序结果表明蜕膜化4 d和敲减TCF3后差异基因主要富集在细胞因子-细胞因子受体通路，GSEA分析验证了这一结果。RT-qPCR结果验证了敲减TCF3调节多种细胞因子的表达，如下调白血病抑制因子（leukemia inhibitory factor，LIF）、白细胞介素-6（interleukin-6，IL-6）、白细胞介素-1β（interleukin-1β，IL1B）、白细胞介素-1受体1（interleukin-1 receptor type 1，IL1R1）和集落刺激因子1（colony stimulating factor 1，CSF1）。结论·下调TCF3表达可能通过调节细胞因子LIF、IL-6、IL1B、IL1R1和CSF1的表达来抑制HESC蜕膜化。

关键词： 人子宫内膜基质细胞; 转录因子3; 蜕膜化; 细胞因子

本文引用格式

卫晓薇 , 田福举 , 刘晓瑞 , 曾维宏 , 陈彩莲 , 林羿 . 敲减TCF3抑制人子宫内膜基质细胞蜕膜化的研究[J]. 上海交通大学学报（医学版）, 2022 , 42(9) : 1247 -1257 . DOI: 10.3969/j.issn.1674-8115.2022.09.011

Abstract

Objective ·To investigate the effect of transcription factor 3 (TCF3) on decidualization of human endometrial stromal cells (HESCs). Methods ·HESC decidualization in vitro was induced by 8-bromoadenosine 3',5'-cyclic adenosine monophosphate (8-Br-cAMP) and medroxy progesterone acetate (MPA). Small interfering RNA (siRNA) against TCF3 was used to construct TCF3-knockdown cell model. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to confirm the knockdown efficiency of TCF3. RT-qPCR and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of decidual markers, including insulin like growth factor binding protein 1 (IGFBP1) and prolactin (PRL). The morphological changes of HESCs after decidualization and after TCF3 knockdown were detected by Alexa Fluor-labeled phalloidin staining. The HESCs were divided into three groups: non-decidualized+NC (transfected with control siRNA) group, decidualized 4 d+NC group, and decidualized 4 d+siTCF3 (transfected with TCF3 siRNA) group, and RNA sequencing was performed. In order to explore the mechanism of TCF3 regulating the decidualization of HESCs, cluster analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and gene set enrichment analysis (GSEA) were conducted in the three groups of cells. qPCR was used to confirm the results of RNA sequencing analysis. Results ·The expression level of TCF3 increased gradually after 2 d and 4 d of decidualization of HESCs. Both ELISA and RT-qPCR results showed that after decidualization for 2 d or 4 d, the expression of decidual markers IGFBP1 and PRL decreased after TCF3 knockdown. Phalloidin staining results showed that after decidualization, the cell morphology changed from a long and slender fusiform structure to a large and round cell shape, while after TCF3 was knockdown, the cell morphology returned to the long fusiform structure. RNA sequencing analysis showed that the differential genes were mainly enriched in the cytokine-cytokine receptor pathway, which was further verified by GSEA analysis. RT-qPCR results confirmed that knockdown of TCF3 down-regulated the expression of several cytokines, such as leukemia inhibitory factor (LIF), interleukin 6 (IL-6), interleukin 1 β (IL1B), interleukin 1 receptor type 1 (IL1R1), and interleukin 1 receptor type 1 (CSF1). Conclusion ·Down-regulation of TCF3 in HESCs inhibits decidualization maybe through regulating the expression of cytokines IL-6, IL1B, IL1R1 and CSF1.

Key words： human endometrial stromal cell (HESC); transcription factor 3 (TCF3); decidualization; cytokine

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